Novel oxime and oxime ether derivatives of 12,14-dichlorodehydroabietic acid: Design, synthesis, and BK channel-opening activity

Article abstract of DOI:10.1016/j.bmcl.2008.10.078

Oxime ether derivatives of the benzylic ketone of 12,14-dichlorodehydroabietic acid (diCl-DHAA, 4b) were synthesised, and their BK channel-opening activity was evaluated in an assay system of CHO-K1 cells expressing hBKα channels. Oxime ether structure on the B ring of diCl-DHAA significantly increased the BK channel-opening activity.

Full text of DOI:10.1016/j.bmcl.2008.10.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6386–6389
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel oxime and oxime ether derivatives of 12,14-dichlorodehydroabietic
acid: Design, synthesis, and BK channel-opening activity
Yong-Mei Cui a, Eriko Yasutomi a, Yuko Otani a, Takashi Yoshinaga b, Katsutoshi Ido b,
Kohei Sawada b, Masatoshi Kawahata c, Kentaro Yamaguchi c, Tomohiko Ohwada a,
*
a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b Tsukuba Research Laboratories, Eisai Co. Ltd, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
c Department of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxime ether derivatives of the benzylic ketone of 12,14-dichlorodehydroabietic acid (diCl-DHAA, 4b)
Received 9 September 2008
Revised 15 October 2008
Accepted 17 October 2008
Available online 22 October 2008
were synthesised, and their BK channel-opening activity was evaluated in an assay system of CHO-K1
cells expressing hBK
a channels. Oxime ether structure on the B ring of diCl-DHAA significantly increased
the BK channel-opening activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
BK channels
Openers
12,14-Dichlorodehydroabietic acid
Oxime
Oxime ether
Large-conductance voltage-gated and calcium-dependent K+
channels (Maxi-K or BK channels) are widely distributed in smooth
muscle, neurons, and many other tissues, and play importantroles in
many physiological events.1 BK channels are uniquely regulated by
changes in both transmembrane potential and intracellular Ca2+ le-
vel,2 and may couple with other ion channels (such as Ca2+ ion chan-
nels)3,4 to serve as a negative feedback pathway controlling ionic
homeostasis, cell excitability, and neuron activity.5 BK channels con-
openers and have been the most widely used pharmacological
tools in investigations of the function of BK channels, as well as
having been lead compounds for the design of several novel syn-
thetic BK openers. Biaryl amines, such as mefenamic and flufen-
amic acids, biarylureas (NS1608), aryloxindoles (BMS-204352),
arylpyrroles (NS-8), and indole-3-carboxylic acid esters (CGS-
7184 and CGS-7181) have also been described and characterized
as BK channel openers.12,13 In addition to these synthetic com-
pounds, a number of naturally derived compounds have been eval-
uated as BK channel openers, including dehydrosoyasaponin-I
(DHS-I),14 L-735334,15 bile acids,16and terpenes such as maxikdiol
(2, Chart 1).17 However, scaffolds for the molecular design of BK
channel openers are still limited and the compounds which
showed the opening activities more potent than NS1619 are still
rare.10,18
Our pioneer work on pimarane compounds, which have an intu-
itive structural similarity to maxikdiol (2), has revealed that pima-
ric acid (3) is a potent BK channel opener.19 Other terpene
analogues, such as abietic acid, sclareol, and methyl pimarate, do
not display significant ability to activate BK channels, despite their
chemical structural similarity. On the other hand, dehydroabietic
acid (DHAA, 4a), which is structurally related to non-aromatic abi-
etic acid, showed weak but definite BK channel-opening activity.20
Chemical modification of DHAA by introduction of halogen atoms
on the benzene ring to afford 12,14-dichlorodehydroabietic acid
(diCl-DHAA, 4b) markedly increased the BK channel-opening activ-
ity.20 All these BK channel openers are assumed to interact with
sist of channel-forming
ranged in tetramers.6 Recent cloning studies have revealed the
presence of multiple splice variants of -subunits and multiple sub-
a-subunits and accessory b-subunits ar-
a
types of b-subunits (b1, b2/b3, and b4).7 The distribution of the b-sub-
units is tissue- and organ-specific (e.g., b1: smooth muscle, b4:
brain).8 Because of the large conductance, significant intracellular
Ca2+ concentration dependency, and potential organ selectivity
due to the b subunits, control of opening of BK channels is a poten-
tially powerful intervention in the modulation of muscle contractil-
ity or neurotransmitter release and hormone secretion. BK channel
openers have emerged as potential targets of drug treatment for
acute stroke, epilepsy, psychoses, erectile dysfunction, arterial
hypertension, asthma, and bladder overactivity.1,9
Several series of compounds have been reported to be BK chan-
nel openers.10 Synthetic benzimidazolone derivatives, represented
by NS004 and NS1619 (1, Chart 1),11 were the pioneer BK channel
* Corresponding author. Tel.: +81 3 5841 4730; fax: +81 3 5841 4735.
E-mail address: ohwada@mol.f.u-tokyo.ac.jp (T. Ohwada).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.078

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6386–6389
6387
1
R
Cl
12
H
N
F C
3
12
O
OH
1
C
C
9
N
OH
2
14
R
Cl
R
14
10
8
A
B
A
B
6
5
H
H
N
O
4
7
H
H
OH
H
CO H
CO H
2
F C
2
CO H
2
3
4a: R1=R2=H
5a-g
4b (diCl-DHAA): R1=R2=Cl
NS1619 (1)
Maxikdiol (2)
Pimaric acid (3)
Chart 1.
the
a
subunit of the BK channels.21 Using DHAA and diCl-DHAA as
formation of 6a is presented in Figure 1.25 Additional molecular
modeling studies provided further insights into the features of
the pharmacophore of the oxime ether openers. In calculations
combined with conformation searching based on Monte Carlo sim-
ulations with OPLS-AA force field and subsequent DFT structure
optimization (B3LYP/6-31G(d)), the energy minimum structure of
the oxime 5a was the E geometry and the energy difference be-
tween E and Z geometries of the oxime is 2.4 kcal/mol. The opti-
mized structure of 5a-E was in good agreement with the X-ray
crystallographic structure of the ester form 6a (Fig. 2). The calcula-
tion also showed that O-methyl derivative 5b favors the E geome-
try of the oxime group, and the calculated energy difference
between E and Z geometries is 2.4 kcal/mol. Because of the pres-
ence of steric repulsion between the oxime moiety and the aro-
matic chloride atom at the 14-position, the oxime moiety is not
conjugated with the aromatic ring and the oxime nitrogen atom
is placed out of the aromatic plane (see Fig. 2).26 This structural
distortion can be represented in terms of the dihedral angle
(\C6C7C8C9); the angles differ in the calculated structures 5a-E
(34.4°) and 5a-Z (47.3°). The former value is well consistent with
that of the crystal structure 6a (39.0°).
a starting point, a number of derivatives, wherein the C ring was
modified by introduction of divergent substituents, have been syn-
thetically prepared and some of them have been characterized as
BK channel openers.20,22 However, modification of the B ring of
diCl-DHAA has been little explored.23
As a part of our studies to find more potent BK channels open-
ers,22,24 we synthesized a series of oxime (5a) and oxime ether
derivatives (5b–g) of the benzylic ketone of diCl-DHAA (4b) and as-
sayed them for BK channel-opening activity. Herein, we present
our finding that the oxime ether structure, particularly when bear-
ing O-short carbon chains, significantly increased the BK channel-
opening activity of diCl-DHAA.
The parent oxime 5a was synthesized as shown in Scheme 1.
Benzylic oxidation of the methyl ester derivative 4c with CrO3 in
AcOH/Ac2O afforded the 7-keto derivative 4d. Treatment of the ke-
tone 4d with hydroxylamine hydrochloride in EtOH and pyridine
under reflux afforded the corresponding E-oxime 6a (anti) as the
sole isolated product. Finally, the methyl ester of 6a was hydro-
lyzed with KOH and 18-crown-6 in methanol to give the desired
carboxylic acid 5a.
We have synthesized oxime ethers bearing various O-alkyl and
phenylalkyl groups, in order to probe the effect of the type and size
of the oximic chains on the BK channel-opening activity. As shown
in Scheme 1, compound 5b was obtained by condensation of ke-
tone intermediate 4d with methoxylamine hydrochloride, fol-
lowed by hydrolysis of the methyl ester with KOH and 18-
crown-6 in methanol. Compounds 5c–g were obtained by O-alkyl-
ation of the oxime 6a with various alkyl bromides and subsequent
basic hydrolysis of the methyl ester, as depicted in Scheme 2.
The exclusive formation of E-structure of the oxime of 6a with
respect to the aromatic ring was unambiguously confirmed by X-
ray crystallographic analysis. An ORTEP plot of the solid state con-
The activities of all the target compounds 5a–g as BK channel
modulators were evaluated by means of automated planar array
patch clamp recording using the 64-well Population Patch Clamp
(PPC) technique.27,28 The BK channel was activated by applying a
step pulse to +100 mV from the holding potential of À90 mV to
CHO-K1 cells expressing hBK
a
channels, and the current amplitude
M) was expressed as per-
in the presence of test compounds (30
l
cent of the drug-free control. The values represent an average of
data obtained from at least eight separate measurements. Stock
solutions of test compounds were prepared in DMSO at a concen-
tration of 10 mM and diluted in buffer solution to give the desired
Cl
Cl
Cl
Cl
Cl
Cl
a
b
Cl
a
Cl
N
N
H
H
H
CO2Me
CO2Me
OH
OR
H
H
CO2H
CO2Me
CO2H
6a
6c-g
4a
4c
4b
R
Cl
b
Cl
Cl
Cl
c
d
Cl
e
e
d
Cl
Cl
c
Cl
N
N
N
OR
O
H
H
H
f
H
CO2H
OR
CO2Me
OR
CO H
CO2Me
2
5c-g
6a R=H
6b R=Me
5a R=H
5b R=Me
4d
g
Scheme 1. Reagents and conditions: (a) Cl2, FeCl3/SiO2, DDQ/SiO2, CCl4, 39%; (b)
TMSCHN2, MeOH–toluene, rt, 90%; (c) CrO3, AcOH/Ac2O, 58%; (d) NH2OR.HCl, EtOH,
Py, reflux, 87–100%; (e) KOH, 18-crown-6, MeOH, reflux, 79–88%.
Scheme 2. Reagents and conditions: (a) RBr, TBAB, 1 N KOH, CH2Cl2, 40 °C, 60–89%;
(b) KOH, 18-crown-6, MeOH, reflux, 43–85%.

6388
Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6386–6389
700
600
500
400
300
200
100
0
compound number
Figure 3. Effects of oxime and oxime ether derivatives 5a–g on hBK
currents (n = 8). Bars represent increase in ionic current (% of control level) in the
presence of test compounds (30 M).
a channel
Figure 1. X-ray crystal structure of the oxime ester 6a.25 Dihedral angle
\C6C7C8C9 = 39.0°.
l
reduced the activity, as compared with the saturated chain deriva-
tive (5f).
Superimposition of the energy-minimized conformations 5a–
5g is shown in Figure 4. The results from a simple overlay of these
structures suggest that the carboxylic acid and tricyclic structures
can be well superposed and can achieve very similar spatial orien-
tations. However, remarkable differences are seen in the direction
and extension of the hydrophobic moieties at the oxime O-alkyl
chain of these compounds. Although the exact binding domain of
these compounds to BK
that the hydrophobic region around C7 is important for the inter-
action with the channel protein subunit. In fact, introduction of
a protein is not known at present, it is clear
a
an O-isopropyl group to the oxime functionality resulted in a dra-
matic increase in BK channel-opening activity as compared with
the oxime itself (5a), the potency being comparable to that of com-
pound 5b (data not shown).
In summary, the oxime structure is a suitable scaffold for
designing novel potent BK-openers. The preliminary structure–
activity relationships of our oxime and oxime ether derivatives
suggested that O-methyl and O-allyl substitutions provide potent
BK channel-opening activity, and compounds with these substitu-
ents were more potent than the potent known BK-channel opener,
NS1619. The BK-channel openers developed in this study might be
candidates for therapeutic application.
Figure 2. Optimized conformations of 5a-E and 5a-Z (unconjugated oxime moiety).
final concentration (the final DMSO concentration was less than
0.4%(v/v)). NS1619 and diCl-DHAA (4b) are included as positive
BK channel openers.
As shown in Figure 3, the oxime 5a (ion current = 242.0 21.2%
of control at 30 lM) was found to be an effective BK opener, with
potency slightly higher than that of the positive compound 4b
(diCl-DHAA, 180.4 11.9%). Introduction of a small saturated sub-
stituent such as methyl (5b, 409.7 31.8%) or small unsaturated
substituents (5c, 566.5 26.3%; 5d, 425.1 42.2%) increased the
potency remarkably; compound 5c (CYM04) was approximately
twice as potent as NS1619 (325.2 28.8%). On the other hand,
introduction of a phenyl-substituted carbon chain had little influ-
ence (5e) or decreased the activity (5f, 5g) compared with the O-
unsubstituted oxime 5a. It is noteworthy that introduction of a ri-
gid alkynyl carbon chain bearing a phenyl group (5g) significantly
Figure 4. Overlay of optimized structures of 5a–5g. Compound 5a: light blue; 5b:
orange; 5c: green; 5d: ochre; 5e: light purple; 5f: dark gray; 5g: petroleum blue.
Hydrogen atoms are omitted for clarity.

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6386–6389
6389
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Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Science, Sports, and Culture
of Japan. Y.-M.C. is grateful for a postdoctoral fellowship for a for-
eign researcher from the Japan Society for the Promotion of Sci-
ence, and also thanks Prof. Masakatsu Shibasaki, Graduate School
of Pharmaceutical Sciences, The University of Tokyo, for valuable
discussions and encouragement in the late stage of this program.
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r
deposition number: CCDC 705340.
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